Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Competition-based proliferation assays in 23 human cancer cell lines infected with the indicated sgRNAs. PDAC, pancreatic ductal adenocarcinoma; RMS, rhabdomyosarcoma. n = 3. (B) Western blot of whole-cell lysates from MOLM-13 cells on day 5 post-infection with the indicated sgRNAs. (C) Competition-based proliferation assay in MOLM-13 cells infected with the indicated sgRNAs. n = 3. (D) Western blot of whole-cell lysates from K562 cells on day 5 post-infection with the indicated sgRNAs. (E) Competition-based proliferation assay in K562 cells infected with the indicated sgRNAs. n = 3. (F) Quantification of the different cell-cycle stages in MOLM-13 cells on day 5 post-infection with the indicated sgRNAs. n = 3. (G) Bioluminescence imaging of NSG mice transplanted with luciferase + /Cas9 + MOLM-13 cells infected with either sgROSA or sgSCP4. (H) Quantification of bioluminescence intensity. n = 3. p value was calculated by unpaired Student’s t-test. *p < 0.05. (I) Western blot of whole-cell lysates from CD34 + cells electroporated with Cas9 loaded with the indicated sgRNAs on day 8 post-electroporation, with culturing in myeloid conditions. (J) qRT-PCR analysis of indels presence in CD34 + cells electroporated with Cas9 loaded with the indicated sgRNAs over the course of culturing in myeloid conditions. The effects of individual sgRNAs for SCP4 were averaged. n = 4. (K) Quantification of the flow cytometry analysis of myeloid differentiation of CD34 + cells electroporated with Cas9 loaded with the indicated sgRNAs on day 8 post-electroporation, with culturing in myeloid conditions. The effects of individual negative controls and sgRNAs for SCP4 were averaged. n = 4. All bar graphs represent the mean ± SEM. All sgRNA experiments were performed in Cas9-expressing cell lines. “e” refers to the exon number targeted by each sgRNA. The indicated sgRNAs were linked to GFP. ROSA, Mock, and NT, negative controls; PCNA and MYC, positive controls. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Infection, Western Blot, Proliferation Assay, Imaging, Luciferase, Electroporation, Quantitative RT-PCR, Flow Cytometry, Expressing

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Relative evolutionary conservation score for each residue from 0 — least conserved to 1 — most conserved. Based on data from . (B) Protein disorder prediction score for each residue from 0 — least disordered to 1 — most disordered. Based on data from . (C) Running average of log 2 fold changes of the CRISPR scan of SCP4 with all the possible sgRNAs. SCP4 protein amino acid numbers are indicated along the x axis. (D) Domain architectures of human SCP4 and the truncated version of SCP4 used in this study. (E) Western blot of whole-cell lysates from MOLM-13 cells stably expressing empty vector or CRISPR-resistant SCP4 236–466 . (F) Competition-based proliferation assay in MOLM-13 cells stably expressing empty vector or CRISPR-resistant SCP4 236–466 infected with the indicated sgRNAs. n = 3. (G) Western blot of whole-cell lysates from MOLM-13 cells stably expressing empty vector (EV; underloaded) or CRISPR-resistant wild type (WT) and catalytic mutants of SCP4. Vertical black dashed lines indicate omitted lanes from the same gel and same exposure. (H) Competition-based proliferation assay in MOLM-13 cells stably expressing empty vector or CRISPR-resistant WT and catalytic mutants of SCP4 infected with the indicated sgRNAs. n = 3. All bar graphs represent the mean ± SEM. ROSA, negative control; PCNA, positive control. See also and .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Residue, CRISPR, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Proliferation Assay, Infection, Negative Control, Positive Control

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Western blot of cytoplasm and nuclear fractions from MOLM-13 cells. (B) Representative FLAG-SCP4 affinity purification western blot analysis for the subsequent mass spectrometry (MS) analysis. Cytoplasm and nucleus fractions from MOLM-13 cells stably expressing empty vector, FLAG-SCP4 WT, or catalytic mutant (D293A). Nuclear fraction was mixed with anti-FLAG M2 agarose overnight. The flow-through was analyzed to ensure efficient binding of the FLAG-tagged constructs (loaded as “unbound”). Following extensive washing, the agarose amount equivalent to the cytoplasm and nucleus fractions loading was boiled in Laemmli buffer and loaded as “FLAG-IP.” The rest was sent for the MS analysis. (C) Venn diagram depicting the overlap between proteins detected by MS and absent in empty vector controls in the 2 independent biological replicates. (D) Total unique peptide counts for SCP4, PDIK1L, and STK35 detected by MS in the 2 independent biological replicates. (E) Domain architectures and homology heat-map of human STK35 and PDIK1L. ATP-BS, ATP-binding site. (F–H) Immunoprecipitation followed by western blotting performed with the indicated antibodies. The whole-cell lysate was prepared from HEK293T 24 h post-transfection with the indicated constructs (F). The nuclear lysates were prepared from the human MOLM-13 cells stably expressing the indicated constructs (G and H). –, empty vector; WT, wild-type FLAG-SCP4; 236–466, FLAG-SCP4 236–466 ; D293A, catalytic mutant FLAG-SCP4 D293A ; IP, immunoprecipitation. Note: degradation bands appear in the WT and D293A input at ~50 kDa and in D293A at ~40 kDa. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Western Blot, Affinity Purification, Mass Spectrometry, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Binding Assay, Construct, Immunoprecipitation, Transfection

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A and B) Western blot of cytoplasm (Cyto) and nucleus (Nucl) fractions of MOLM-13 cells stably expressing HA-PDIK1L (A) or HA-STK35 (B) on day 5 post-infection with the indicated sgRNAs. Shown is a representative experiment of 3 independent biological replicates. (C) Schematics of phosphorylation sites reported in the publicly available phospho-proteomics datasets on STK35 and PDK1L relative to their domain architectures ( ; ). aa #, amino acid number; P, phosphorylation; ATP-BS, ATP-binding site. (D) Phosphorylated peptides assayed for SCP4 phosphatase activity in vitro . (E) Recombinant SCP4 protein purity as assessed by SDS-PAGE and Coomassie blue staining. His-SUMO-(TEV)-SCP4 was expressed in BL21 (DE3) cells and purified by affinity, anion exchange, and gel filtration chromatography. Molecular weight markers are shown for reference. (F and G) Phosphatase activity of SCP4 against the indicated peptides plotted for kinetic fitting. Each measurement was conducted in triplicate with standard deviations shown as error bars. (H) Competition-based proliferation assay in MOLM-13 cells stably expressing EV or the CR HA-PDIK1L or HA-STK35 constructs harboring the indicated amino acid substitutions infected with the indicated sgRNAs. n = 3. (I) Western blot of whole-cell lysates from MOLM-13 cells stably expressing EV or CR HA-PDIK1L or HA-STK35 constructs harboring the indicated amino acid substitutions. All bar graphs represent the mean ± SEM. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Western Blot, Stable Transfection, Expressing, Infection, Phospho-proteomics, Binding Assay, Activity Assay, In Vitro, Recombinant, SDS Page, Staining, Purification, Filtration, Chromatography, Molecular Weight, Proliferation Assay, Construct

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: (A) Venn diagram depicting the overlap between statistically significant downregulated genes in MOLM-13 cells upon SCP4 knockout and STK35/PDIK1L double knockout. DeSeq2 (n = 4). (B) Ontology analysis of overlapping statistically significant downregulated genes in MOLM-13 cells upon both SCP4 knockout and STK35/PDIK1L double knockout. (C) Selected statistically significant downregulated genes in MOLM-13 cells upon both SCP4 knockout and STK35/PDIK1L double knockout and the amino acids they are involved in biosynthesis or transport of. (D) The correlation between log2 fold changes in the levels of selected metabolites upon SCP4 knockout and STK35/PDIK1L double knockout relative to negative control as measured by the MS analysis. Every dot represents the mean ± SEM (n = 6). Amino acids are in red, and there are a few unchanged metabolites in black for reference. Shown is a representative experiment of 3 independent biological replicates. (E) Model. See also .

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Knock-Out, Double Knockout, Negative Control

Journal: Cell reports

Article Title: SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

doi: 10.1016/j.celrep.2021.110233

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used in this study included SCP4 ( CTDSPL2 ) (CST, # 6932, 1:500), FLAG (Sigma Aldrich, F1804, 1:10,000), HA-HRP (Sigma Aldrich, clone 3F10, 1:10,000), H3K4me3 (Sigma Aldrich, 07–473, 1:1,000), β-Actin-HRP (Sigma A3854-200UL; 1:50,000).

Techniques: Virus, Recombinant, Lysis, Plasmid Preparation, Magnetic Beads, Cloning, Purification, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Gel Extraction, Ligation, Sample Prep, Mass Spectrometry, Software

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: Flow chart depicting steps to assemble the filtration units and the methodology to be followed for isolating gDNA using the PES membrane. Note that the methodology described requires no centrifugation steps. RT, room temperature; TE, Tris-EDTA.

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Filtration, Membrane, Centrifugation

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: Agarose gel electrophoresis of gDNA samples prepared using different PES membranes or the QIAamp commercial kit. A) Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B) Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; and lane 3: Sterlitech). C) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb). Representative of a minimum of 4 independent experiments.

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Agarose Gel Electrophoresis, Extraction, Membrane, Marker

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: PCR of different HLA targets using gDNA prepared using the PES membrane or QIAamp kit. Buffy coat from the same blood sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (A) or QIAamp DNA Blood Mini Kit from Qiagen (B). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter. PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1 and 2: HLA-A Exons 2 and 3; lanes 3 and 4: HLA-B Exons 2 and 3; lanes 5 and 6: HLA-C Exons 2 and 3; lanes 7 and 10: HLA-DPB1 Exons 3 and 2, respectively; lanes 8 and 11: HLA-DQB1 Exons 3 and 2, respectively; lanes 9 and 12: HLA-DRB1 Exons 3 and 2, respectively. M, DNA MW marker (250 bp–10 kb).

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Membrane, Extraction, Amplification, Biomarker Discovery, Isolation, Agarose Gel Electrophoresis, Marker

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: PCR of different HLA targets using gDNA prepared using PES membranes from different suppliers. Buffy coat from different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers: EMD Millipore (A), Pall (B), and Sterlitech (C). Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter. PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lane 1: HLA-A Exon 2; lane 2: HLA-A Exon 3; lane 3: HLA-B Exon 2; lane 4: HLA-B Exon 3; lane 5: HLA-C Exon 2; lane 6: HLA-C Exon 3; lane 7: HLA-DPB1 Exon 3; lane 8: HLA-DQB1 Exon 3; lane 9: HLA-DRB1 Exon 3; lane 10: HLA-DPB1 Exon 2; lane 11: HLA-DQB1 Exon 2; lane 12: HLA-DRB1 Exon 2. M, DNA MW marker (250 bp–10 kb).

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Extraction, Membrane, Amplification, Biomarker Discovery, Isolation, Agarose Gel Electrophoresis, Marker

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: PCR of different HLA targets using gDNA prepared by PES membranes with different pore sizes. Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm (A) or 0.45 µm (B) PES membrane from EMD Millipore. Different targets of HLA loci were PCR amplified, as described in Validation of the gDNA isolation method using the PES membrane filter. PCR amplicons were analyzed on 2.5% agarose gel electrophoresis. Representative of a minimum of 4 independent experiments. Lanes 1–6: HLA-A Exon 2, HLA-A Exon 3, HLA-B Exon 2, HLA-B Exon 3, HLA-C Exon 2, and HLA-C Exon 3; lanes 7–9: Exon 3 genes of HLA-DPB1, DQB1, and DRB1; lanes 10–12: Exon 2 genes of DPB1, DQB1, and DRB1. M, DNA MW marker (250 bp–10 kb).

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Extraction, Membrane, Amplification, Biomarker Discovery, Isolation, Agarose Gel Electrophoresis, Marker

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

doi: 10.7171/jbt.18-2902-001

Figure Lengend Snippet: Agarose gel electrophoresis of gDNA samples digested with either HindIII or EcoRI. DNAs digested with HindIII (A–C); EcoR1 digested gDNAs (D–F). A, D) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either 0.22 µm PES membrane from EMD Millipore (lane 1) or QIAamp DNA Blood Mini Kit from Qiagen (lane 2). B, E) Buffy coat from 2 different samples was lysed independently, pooled together, and equally distributed and processed for gDNA extraction using the 0.22 µm PES membrane from different suppliers (lane 1: EMD Millipore; lane 2: Pall; lane 3: Sterlitech). C, F) Buffy coat from the same sample was equally distributed and processed for gDNA extraction using either the 0.22 µm (lane 1) or 0.45 µm (lane 2) PES membrane from EMD Millipore. M, DNA MW marker (75 bp–20 kb).

Article Snippet: Estimation of the gDNA yield DNA concentrations of different samples were estimated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Agarose Gel Electrophoresis, Extraction, Membrane, Marker

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: TGF-β1 expression and exosome production are increased in UUO-induced renal fibrosis models. (A-C) Representative micrographs of immunohistochemical staining (A) and quantitative data (B,C) show TGF-β1 and Collagen I expression at different time points after UUO. Scales bars = 50μm. * p < 0.05 versus sham, # p < 0.05 versus UUO-3d (n = 6). (D-F) Representative micrographs of Masson staining and Sirius red staining (D) and quantitative data (E, F) show collagen fiber accumulation after UUO. Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus UUO-3d (n = 6). (G, H) Representative western blot (G) and quantitative data (H) on TGF-β1, fibronectin and exosomal-specific proteins CD63 and TSG101 after UUO. Numbers (1 to 3) indicate each individual animal in the given group. * p < 0.05 versus sham, # p < 0.05 versus UUO-3d (n = 6). (I) Transmission electron microscopy (TEM) shows the exosomes and microvesicles released by renal tubular epithelial cells after UUO. Scales bars = 300 nm. (J) Double immunofluorescence staining (green for CD63 and red for TSG101) demonstrates the generation of exosomes predominantly in tubular epithelial cells after UUO. Scales bars = 5 μm. (K, L) Quantitative determination of CD63- and TSG101-positive renal tubules. Data were obtained from 3 images per mouse, with 6 mice per group. * p < 0.05 versus sham, # p < 0.05 versus UUO-3d.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Transmission Assay, Electron Microscopy, Double Immunofluorescence Staining

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: TGF-β1 promotes the secretion of exosomes by renal tubular epithelial cells and activates fibroblasts in vitro . (A) Schematic diagram of experimental process. Exosomes from NRK-52E cells treated without (Ctrl-Exos) or with TGF-β1 (TGFβ1-Exos) were extracted and incubated with NRK-49F cells. (B,C) DLS and NTA of exosomes from NRK-52E cells. (D) TEM image of exosomes isolated from NRK-52E cells. Scale bar = 100 nm. (E, F) Representative western blot (E) and quantitative data (F) of CD63 as an exosome marker in exosomes from TGF-β1- or GW4869-treated NRK-52E cells. Numbers (1 to 3) indicate each independent treatment in the given group. * p < 0.05 versus Ctrl-Exos, # p < 0.05 versus 5 ng/ml TGFβ1-Exos, & p < 0.05 versus 15 ng/ml TGFβ1-Exos (n = 3). (G) Fluorescent staining image of PKH-67-labeled NRK-52E cells. Scales bars=50 μm. (H) Fluorescent staining image of NRK-52E cell-derived exosomes taken up by NRK-49F cells. Scales bars=10 μm. (I, K) Representative western blot (I) and quantitative data (K) of α-SMA and PCNA in NRK-49F cells incubated with exosomes from TGF-β1- or GW4869-treated NRK-52E cells. Numbers (1 to 3) indicate each independent treatment in the giving group. * p < 0.05 versus Ctrl-Exos, # p < 0.05 versus 5 ng/ml TGFβ1-Exos, & p < 0.05 versus 15 ng/ml TGFβ1-Exos (n = 3). (J) Proliferation rate of NRK-49F cells incubated with NRK-52E cell-derived exosomes measured by CCK-8. * p < 0.05 versus Ctrl-Exos, # p < 0.05 versus 5 ng/ml TGFβ1-Exos, & p < 0.05 versus 15 ng/mL TGFβ1-Exos (n = 3). (L-N) Double immunofluorescence staining (green for Col-I and red for fibronectin) demonstrates the expression of Col-I and fibronectin in NRK-49F cells incubated with NRK-52E cell-derived exosomes. Scales bars=50 μm. * p < 0.05 versus Ctrl-Exos, # p < 0.05 versus 5 ng/ml TGFβ1-Exos, & p < 0.05 versus 15 ng/mL TGFβ1-Exos.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: In Vitro, Incubation, Isolation, Western Blot, Marker, Staining, Labeling, Derivative Assay, CCK-8 Assay, Double Immunofluorescence Staining, Expressing

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Tubular cell-derived exosomes promote renal fibrosis in vivo . (A) Experimental design. TGFβ1-Exos or Ctrl-Exos from NRK-52E cells were injected into UUO mice by tail vein injection at 1, 3 and 5 days. (B) Representative images show the presence of PKH-67-labeled TGFβ1-Exos isolated from NRK-52E cells in mouse kidneys after intravenous injection. Kidney sections were examined at 24 h after injection. Arrows indicate PKH-67 labeled exosomes (green). Scales bars=50 μm. (C, D) Representative western blot (C) and quantitative data (D) of TSG101, CD63 and fibronectin in UUO kidneys injected with Ctrl-Exos or TGFβ1-Exos. Numbers (1 to 3) indicate each individual animal in the given group. * p < 0.05 versus sham, # p < 0.05 versus UUO7d+Ctrl-Exo (n = 6). (E, F) Representative western blot (E) and quantitative data (F) of fibronectin, Fsp-1 and PCNA in UUO kidneys injected with Ctrl-Exos or TGFβ1-Exos. Numbers (1 to 3) indicate each individual animal in the given group. * p < 0.05 versus sham, # p < 0.05 versus UUO7d+Ctrl-Exo (n = 6). (G, J) Treble immunofluorescence staining (G) and quantitative data (J) demonstrate the expression of E-cad, α-SMA and fibronectin in UUO kidneys injected with Ctrl-Exos or TGFβ1-Exos. Scales bars=50μm. * p < 0.05 versus sham, # p < 0.05 versus UUO7d+Ctrl-Exo. (H, I, K, L) Representative micrographs of Col-I immunohistochemical staining, Masson staining, Sirius red staining (H) and quantitative data (I, K, L) show fibronectin and collagen deposition in UUO kidneys injected with Ctrl-Exos or TGFβ1-Exos. Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus UUO7d+Ctrl-Exo.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Derivative Assay, In Vivo, Injection, Labeling, Isolation, Western Blot, Immunofluorescence, Staining, Expressing, Immunohistochemical staining

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Rab27a knockout inhibits exosome secretion and alleviates UUO-induced renal fibrosis in vivo . (A) Rab27a -/- mouse. (B, C) Rab27a knockout was confirmed by PCR screening (targeted allele: 476 bp) (B) and sequencing confirmation (C) . (D, E) Representative western blot (D) and quantitative data (E) of CD63 in Rab27a knockout kidneys after UUO (n = 6). * p < 0.05 versus sham, # p < 0.05 versus WT+UUO-7d. (F, I) Representative western blot (F) and quantitative data (I) of Fsp-1, PCNA and α-SMA in Rab27a knockout kidneys after UUO (n = 6). * p < 0.05 versus sham, # p < 0.05 versus WT+UUO-7d. (H, J) Representative immunofluorescence micrographs (J) and quantitative data (H) show Fsp-1 expression (n = 6). Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus WT+UUO-7d. (G, K-M) Masson staining, Col-I immunohistochemical staining and fibronectin immunofluorescence staining indicated the deposition of collagens and fibronectin. Representative micrographs (G) and quantitative data (K-M) are presented (n = 6). Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus WT+UUO-7d.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Knock-Out, In Vivo, Sequencing, Western Blot, Immunofluorescence, Expressing, Staining, Immunohistochemical staining

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Tubule cell-derived exosomal miR-21 promotes fibroblast activation in vitro . (A) NRK-52E-delivered exosomal miRNA sequencing after TGF-β1 (15 ng/ml) treatment. (B) Experimental design. Concentration of TGF-β1, 15 ng/ml. (C, G-I) Representative immunofluorescence micrographs (C) and quantitative data (G-I) show Col-I, fibronectin and α-SMA expression in NRK-49F cells after stimulation with NRK-52E-delivered exosomes. Scales bars=50 μm. * p < 0.05 versus sham, # p < 0.05 versus Ctrl. (D) PCR detection of miR-21 in exosomes and NRK-52E cells after TGF-β1 treatment (n = 3). * p < 0.05 versus sham in cells, # p < 0.05 versus sham in exosomes. (E) PCR detection of miR-21 in NRK-52E-delivered exosomes after miR-21 mimic or inhibitor transfection (n = 3). * p < 0.05 versus sham, # p < 0.05 versus Ctrl. (F) Proliferation of NRK-49F cells after stimulation with exosomes from NRK-52E cells (n = 3). * p < 0.05 versus sham, # p < 0.05 versus Ctrl.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Derivative Assay, Activation Assay, In Vitro, Sequencing, Concentration Assay, Immunofluorescence, Expressing, Transfection

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Tubule cell-derived exosomal miR-21 mediates fibroblast activation through the PTEN/AKT pathway in vitro . (A) Experimental design. Concentration of TGF-β1, 15 ng/ml. (B) PTEN mRNA level after siPTEN treatment. NS, no significant difference versus Ctrl, # p < 0.05 versus Ctrl. (C) Proliferation of NRK-49F cells after miR-21 inhibitor or siPTEN transfection (n = 3). # p < 0.05. (D-G) Representative immunofluorescence micrographs (G) and quantitative data (D-F) show Col-I, fibronectin and α-SMA expression in NRK-49F cells after miR-21 inhibitor or siPTEN transfection (n = 3). Scales bars=50 μm. # p < 0.05. (H-K) Representative western blot (H) and quantitative data (I-K) show PTEN, Akt, p-Akt and PCNA expression in NRK-49F cells after miR-21 inhibitor or siPTEN transfection (n = 3). # p < 0.05.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Derivative Assay, Activation Assay, In Vitro, Concentration Assay, Transfection, Immunofluorescence, Expressing, Western Blot

Journal: Theranostics

Article Title: Exosomal miR-21 from tubular cells contributes to renal fibrosis by activating fibroblasts via targeting PTEN in obstructed kidneys

doi: 10.7150/thno.62820

Figure Lengend Snippet: Tubular cell-derived exosomal miR-21 promotes renal fibrosis through the PTEN/AKT pathway in vivo . (A-D) Representative immunofluorescence micrographs (A) and quantitative data (B-D) show Col-I, fibronectin and α-SMA expression in mouse kidneys after intravenous exosome injection (n = 6). Scales bars=50 μm. * p < 0.05 versus Ctrl-Exo, # p < 0.05 versus TGFβ1-Exo. (E-I) Representative western blot (F) and quantitative data (E, G-I) show Fsp-1, PCNA, PTEN, Akt and p-Akt expression in mouse kidneys after intravenous exosome injection (n = 6). * p < 0.05 versus Ctrl-Exo, # p < 0.05 versus TGFβ1-Exo.

Article Snippet: The primary antibodies were as follows: TGF-β1 (Sigma, SAB4502954), CD63 (Affinity Biosciences, AF5117), TSG-101 (Abcam, ab125011), Fibronectin (Proteintech Group, 15613-1-AP), α-SMA (Boster Biological Technology, BM0002), Collagen I (Novus Biologicals, NB600-408), E-cadherin (Abcam, ab76319), Fsp-1 (Cell Signaling Technology, 13018), PCNA (Abcam, ab92552), PTEN (Cell Signaling Technology, 9559), and p-Akt (Abcam, ab18785).

Techniques: Derivative Assay, In Vivo, Immunofluorescence, Expressing, Injection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Reduced NMNAT1 levels in db/db mice and human renal fibrosis of DN. ( a ) Immunolocalization of NMNAT1 in the kidneys of nondiabetic control mice (db/m) and diabetic mice (db/db) at 32 weeks of age. The protein expression in the control and diabetic kidneys was examined through immunohistochemistry; representative images are shown. Positive protein expression was stained brown by a 3,3′-diaminobenzidine agent. Scale bar, 50 µm; N = 7. Results of immunohistochemical scoring assessing whole kidneys are expressed as the mean ± standard error of the mean (SEM). * p < 0.05. In addition, we have scored the Nmnat1 levels in tubular and glomerular areas separately. All data are depicted as the mean ± SEM. Horizontal bars denote statistically significant differences between the two groups. * p < 0.05. ( b ) Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of the renal mRNA of NMNAT1, NMNAT2, and NMNAT3. The kidney tissue specimens for RT–PCR were derived from db/m and db/db mice at 32 weeks of age. N = 7 mice per group. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. All data are depicted as the mean ± SEM. Horizontal bars denote statistically significant differences between the two groups. * p < 0.05. ( c ) Masson’s trichrome and NMNAT1 immunostaining in human kidneys. Representative photomicrographs of needle renal biopsy specimens from patients with DN with mild or severe fibrosis. The relationship between the Masson’s trichrome stain-positive area and the immunostaining intensity for NMNAT1 in renal biopsy specimens from patients with DN (N = 11). Pearson’s correlation analysis was used to calculate r and p values. Bars; 50 nm.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Control, Expressing, Immunohistochemistry, Staining, Immunohistochemical staining, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Clinical data of patients with diabetic nephropathy. eGFR, estimated glomerular filtration rate; HbA1c, glycosylated hemoglobin.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Filtration

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Effects of Nmnat1 CKO on urinary albuminuria. ( a ) Nmnat1 immunofluorescence intensity levels in each mouse group. Kidney tissue specimens obtained from each 32-week-old mouse were stained using immunofluorescence for Nmnat1 (green) and AQP1 (red). ( b ) The panel shows the results of the quantitative analysis of Nmnat1 fluorescence intensity. Scale bar, 50 µm; N = 7. ( c ) Real-time quantitative reverse transcription–PCR analysis of renal mRNA of Nmnat1 (N = 7) in control and CKO mice at 32 weeks of age. ( d ) Temporal changes in the mean plasma glucose concentrations in mice from each group. We assessed the mice at 8, 16, 24, and 32 weeks of age. N = 7 mice per group. ( e ) Changes in mouse body weight from 8 to 32 weeks of age. N = 7 mice per group. Horizontal bars indicate statistically significant differences in each group. * p < 0.05. ( f ) Serum creatine levels in each mouse at 32 weeks of age. N = 6 mice per group. ( g ) Urinary albumin excretion in each mouse at 32 weeks of age. N = 6 mice per group. ( h ) Sodium dodecyl-sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of mouse urine samples. Urine samples of mice from each group at 32 weeks of age was tested using 15% SDS–PAGE before staining with Coomassie blue. N = 2 mice per group. AQP1, anti-aquaporin-1; Nmnat1 , nicotinamide mononucleotide adenylyl transferase1; PCR, polymerase chain reaction; Cont, control; CKO, conditional knockout. ( i ) Representative histological findings of the kidneys of control and CKO mice via Masson’s trichrome staining. Scale bar = 500 nm. Percentage of renal fibrosis area (N = 20 mice per group). Horizontal bars indicate statistically significant differences in each group. * p < 0.05. ( j ) Renal tissue concentrations of NAD + metabolites, NAM, NMN, and NAD at 32 weeks of age in the control and CKO groups (N = 7). Statistical significance between each group is represented by a horizontal bar. * p < 0.05.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunofluorescence, Staining, Fluorescence, Reverse Transcription, Control, Clinical Proteomics, Polyacrylamide Gel Electrophoresis, SDS Page, Polymerase Chain Reaction, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Renal phenotypes in Nmnat1 CKO mice. ( a ) Representative images of albumin staining in each mouse. Arrows denote albumin cast in CKO mice. The right panel shows the relative staining intensity. N = 7 mice per group. ( b ) Changes in albumin uptake-related molecules in Pck1 CKO mice. The kidney tissue specimens for RT–PCR were derived from CKO and control mice at 32 weeks of age. N = 7 mice per group. Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of renal mRNA of megalin, cubilin, and amnionless. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. All data are depicted as the mean ± standard error of the mean. Horizontal bars denote statistically significant differences between the two groups. * p < 0.05. ( c ) Representative immunofluorescence double staining of TUNEL (green) and AQP1 (red, proximal tubules) in kidney tissues (N = 7). Scale bars, 50 µm. The kidney tissue specimens were derived from Cont and CKO mice at 32 weeks of age. White arrows denote apoptotic tubular cells.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Control, Reverse Transcription, Polymerase Chain Reaction, Immunofluorescence, Double Staining, TUNEL Assay

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Mitoribosome excess in Nmnat1 CKO mice. ( a ) Representative photomicrographs showing TGF-β immunostaining in each group. The bar graph represents the quantitative analysis of TGF-β staining. N = 7 mice per group. Scale bar = 100 μm. ( b ) Representative photomicrographs indicate collagen IV immunostaining in each group. The bar graph shows the quantitative analysis of collagen IV-stained areas. N = 7 mice per group. Scale bar = 100 μm. Kidney tissue specimens for immunostaining were derived from the four 32-week-old mouse groups. All data are depicted as mean ± standard error of the mean. Horizontal bars indicate statistically significant differences in each group. * p < 0.05. ( c ) Representative electron micrograph in each group. Scale bar = 500 nm. Blue squares indicate the enlarged regions. Expanded images are also presented. Blue arrowheads indicate mitoribosomes. Scale bar = 500 nm. Illustration depicts mitoribosome excess in CKO mice. The number of mitoribosomes was intact in Cont mice. For electron microscopy, the kidney tissue specimens were embedded into Epon epoxy resin. Electron micrographs of 10 proximal tubules (PTs) per kidney were randomly obtained for each mouse to evaluate the morphometry of PTs. The red symbols represent mitoribosomes. ( d ) Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of the renal mRNA of CRIF1, a mitoribosomal synthesis regulator. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. The kidney tissue specimens for RT–PCR were obtained from CKO and control mice at 32 weeks of age. N = 7 mice per group. ( e ) Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of the renal mRNA of intracellular organelle markers. MRPL13 and MRPS15 are mitoribosomal proteins, VDAC is a mitochondrial protein, and Lamin B is a nuclear protein. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. For RT–PCR, the kidney tissue specimens were obtained from CKO and control mice at 32 weeks of age. N = 7 mice per group.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunostaining, Staining, Derivative Assay, Electron Microscopy, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: CRIF1 gene regulation by HIC1 and SP1. ( a ) Localization and nucleotide sequence in the murine CRIF1 promoter region. The red characters represent the effects of Nmnat1 deficiency–mediated by two putative HIC1-responsive element (HiREs) on CRIF1 upregulation. The transcription start sites are indicated in gray. ( b ) The schematic diagram describes seven deletion mutants in the CRIF1 promoter sequences (−1955, −1263, −813, −649, −496, −128, and −15) that were cloned upstream from a luciferase reporter gene. The bar graphs present the results of transient transfection of cultured proximal tubules (PTs), illustrating the promoter activities associated with each deletion. Luciferase activity is shown relative to that of the −1955 Luc vector in the control vector-transfected cells. Values are expressed as the mean ± standard error of the mean (SEM). * p < 0.05 vs. each Luc transfected PTs (N = 3 independent experiments). ( c ) Mutation analysis of CRIF1 promoter activity in cultured PT cells. −649 Luc, WT Nmnat1 promoter; M1, proximal HiRE mutation; M2, distal HiRE mutation; M3, mutation in both HiREs corresponding to the HIC1 binding sites. * p < 0.05 vs. −649 Luc in control cells (N = 3 independent experiments). ( d ) Representative photomicrographs showing HIC1 immunostaining in each group. The bar graph illustrates the results of quantitative analysis. N = 7 mice per group. Light micrograph; scale bar = 100 μm. Kidney tissue specimens for immunostaining were obtained from four mouse groups at 32 weeks of age. All data are shown as mean ± SEM. Horizontal bars indicate statistically significant differences in each group. * p < 0.05. ( e ) Schematic representation of the murine CRIF1 gene and promoter. The solid boxes indicate the binding of HiRE to HIC1 and binding of GC box to Sp1, highlighted in red and yellow, respectively. Cont, control; CKO, conditional knockout; CRIF1, CR6-interacting factor 1; Dmrt1, double sex and mab-3 related transcription factor 1; Egr2, early growth response protein 2; Nr5A2, nuclear receptor subfamily 5 group A member 2; HIC1, HIC ZBTB transcriptional repressor 1; STAT2, signal transducer and activator of transcription 2; Sp1, specificity protein 1; Luc, luciferase; Nmnat , nicotinamide mononucleotide adenylyl transferase; DN, diabetic nephropathy.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Sequencing, Clone Assay, Luciferase, Transfection, Cell Culture, Activity Assay, Plasmid Preparation, Control, Mutagenesis, Binding Assay, Immunostaining, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Nmnat1 Deficiency Causes Mitoribosome Excess in Diabetic Nephropathy Mediated by Transcriptional Repressor HIC1

doi: 10.3390/ijms25126384

Figure Lengend Snippet: Mitoribosomal dysfunction and mitochondrial dysfunction in CKO mice. ( a ) Western blot analysis of renal protein levels of OXPHOS subunits encoded by nDNA and mtDNA. The kidney tissue specimens were obtained from CKO and control mice at 32 weeks of age. The results of a representative experiment among the three performed are shown. All data are indicated as the mean ± standard error of the mean (SEM). Horizontal bars denote statistically significant differences between the two groups. * p < 0.05. The bar graph in the lower panels illustrates the quantification of the band intensity. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. N = 3 mice per group. ( b ) Real-time quantitative RT–PCR analysis of the renal mRNA of mitoribosomal translational regulators. Glyceraldehyde 3-phosphate dehydrogenase was used as a control. For RT–PCR, the kidney tissue specimens were obtained from CKO and control mice at 32 weeks of age. N = 7 mice per group. ( c ) Illustration depicting the dysfunctional mitoribosomes and their concomitant mitochondrial dysfunction in CKO mice. These functions are intact in control mice. ( d ) The oxygen consumption rate (OCR) of TECs isolated from CKO and control mice was measured using a Seahorse XF-24 flux analyzer. N = 3. ( e ) The ratio of red/green fluorescence of JC-1 in TECs isolated from CKO and control mice was used as a measure of mitochondrial membrane potential. N = 7. ( f ) Fluorescence of MitoSox in TECs isolated from CKO and control mice as a measure of mitochondrial levels of reactive oxygen species. N = 7. ( g ) ATP content in TECs isolated from CKO and control mice. N = 7. All data are presented as mean ± SEM. Horizontal bars indicate statistically significant differences between the two groups. * p < 0.05. ( h ) Scheme depicting the new mitoribosome excess-mediated mechanism of renal profibrotic changes and mitochondrial dysfunction in CKO mice. The downregulation of Nmnat1 expression decreased HIC1 expression, resulting in increased CRIF1 expression and ultimately mitoribosome excess. The upregulation of mitoribosome excess leads to mitoribosomal dysfunction, deposition of collagen IV in addition to OXPHOS impairment and tubular mitochondrial dysfunction. MRPL13, mitochondrial ribosomal protein L13; MRPS15, mitochondrial ribosomal protein S15; VDAC, voltage-dependent anion channel; Cont, control; CKO, conditional knockout; OXPHOS, oxidative phosphorylation; ND1, NADH-ubiquinone oxidoreductase chain 1; NDUFA9, NADH: ubiquinone oxidoreductase subunit A9; FP, fluorescent protein; Cyto b, cytochrome b; UQCRC2, ubiquinol-cytochrome c reductase core protein 2; COX1, cytochrome c oxidase 1; ATP8, adenosine triphosphate 8; ATP5A1, ATP synthase F1 subunit alpha; mtIF3, mitochondrial translational initiation factor 3; mtEFTu, mitochondrial elongation factor EFTu; mtRRF, mitochondrial ribosome recycling factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CCCP, carbonyl cyanide m-chlorophenyl hydrazone.

Article Snippet: We conducted dual labeling by incubating overnight 5-μm thick cryostat kidney sections mixed with two primary antibodies, namely, rabbit polyclonal anti- Nmnat1 (1:50, 11399-1AP, Proteintech, Chicago, IL, USA) and mouse polyclonal anti-aquaporin-1 (1:100, B-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Isolation, Fluorescence, Membrane, Expressing, Knock-Out, Phospho-proteomics

Journal: Nature

Article Title: The Molecular Basis of Tight Nuclear Tethering and Inactivation of cGAS

doi: 10.1038/s41586-020-2749-z

Figure Lengend Snippet: a. Surface plasmon resonance (SPR) showing that full length human cGAS binds to nucleosomes purified from HEK 293T cells with nanomolar affinity. b. Surface plasmon resonance (SPR) showing that human cGAS catalytic domain binds to reconstituted nucleosomes with nanomolar affinity. c-d. Bio-Layer interferometry binding studies of full length human cGAS and its catalytic domain with nucleosomes (HEK 293T). e-f. SPR binding studies show that full length mouse cGAS and its catalytic domain bind nucleosomes (HEK 293T) with nanomolar affinities. g-h . Bio-Layer interferometry binding studies of full length mouse cGAS and its catalytic domain with nucleosomes (HEK 293T). i. Gel filtration chromatography (top panel) and SDS-PAGE (lower panel) analyses of 45 bp ISD dsDNA, nucleosome and cGAS mixture show that the nucleosome efficiently competes with dsDNA to bind cGAS. j. Agarose gel electrophoresis of the salmon sperm DNA used in cGAS activity assays. k. cGAS activity assays by ion exchange chromatography show that oligonucleosome binding potently inhibits the activity of cGAS and ligand free cGAS can be activated by the cGAS-oligonucleosome complex.

Article Snippet: For mouse cGAS catalytic domain, 2.5 μM protein was incubated with 0.2 mg/ml salmon sperm DNA (Invitrogen, 15632-011) for 1 h at 37 °C.

Techniques: SPR Assay, Purification, Binding Assay, Filtration, Chromatography, SDS Page, Agarose Gel Electrophoresis, Activity Assay, Ion Exchange Chromatography

Journal: Nature

Article Title: The Molecular Basis of Tight Nuclear Tethering and Inactivation of cGAS

doi: 10.1038/s41586-020-2749-z

Figure Lengend Snippet: a - f. The density maps (grey mesh) of histones H2A, H2B, H3, H4, part of mouse cGAS catalytic domain, and the Widom 601 nucleosome positioning sequence DNA contoured at 3σ. The protein and DNA structures fitted into the density map are shown by the stick models.

Article Snippet: For mouse cGAS catalytic domain, 2.5 μM protein was incubated with 0.2 mg/ml salmon sperm DNA (Invitrogen, 15632-011) for 1 h at 37 °C.

Techniques: Sequencing

Journal: Nature

Article Title: The Molecular Basis of Tight Nuclear Tethering and Inactivation of cGAS

doi: 10.1038/s41586-020-2749-z

Figure Lengend Snippet: a . cGAS binds to the acidic patch of the nucleosome. cGAS is shown by the green ribbons. The histone core is shown by the surface representation colored according to surface electrostatic potential. Positively charged surface is colored in blue and negatively charged surface in red. b . Interactions between cGAS (green) and histone H2A (cyan) and H2B (slate). c . Superposition of the cGAS-dsDNA and the cGAS-nucleosome complex structures shows that the second DNA binding site (site B) of cGAS mediates its interactions with the nucleosome. d . Two overlapping sets of residues are involved in nucleosome and DNA binding by cGAS.

Article Snippet: For mouse cGAS catalytic domain, 2.5 μM protein was incubated with 0.2 mg/ml salmon sperm DNA (Invitrogen, 15632-011) for 1 h at 37 °C.

Techniques: Binding Assay

Journal: Nature

Article Title: The Molecular Basis of Tight Nuclear Tethering and Inactivation of cGAS

doi: 10.1038/s41586-020-2749-z

Figure Lengend Snippet: a . Polyacrylamide gel electrophoretic mobility shift assay (EMSA) shows that mutations at the cGAS-nucleosome interface affect nucleosome binding by mcGAS. In this assay, mcGAS catalytic domain was mixed with nucleosome at molar ratio of 3:1. b . Binding affinities of human cGAS mutants to the nucleosome and the relative catalytic activities of human and mouse cGAS mutants. c . Agarose gel electrophoretic mobility shift assay (EMSA) shows that mutations at the cGAS-nucleosome interface affect dsDNA binding. In this assay, mcGAS catalytic domain was mixed with 45 bp dsDNA at molar ratio of 20:1 d. IFN-β luciferase reporter assays show that mutations of mcGAS affect signaling in HEK 293T cells. Luciferase reporter signals from the cells transfected with 0.004 ng cGAS and 0.4 ng STING are indicated by the orange bars, from cells transfected with 0.001 ng cGAS and 0.4 ng STING by the cyan bars, from cells transfected with 0.004 ng cGAS, 0.4 ng STING, or the vector control by the green, brown and purple bars, respectively. e. cGAMP levels in HEK 293T cells transfected with mcGAS mutants. The cells were transfected with indicated mcGAS plasmids for 24 hours and stimulated with lipofactamine alone or lipofactamine plus salmon sperm DNA, followed by cGAMP assays from the cell lysates. f . cGAMP levels in HEK 293T cells transfected with hcGAS mutants with and without DNA stimulation. In d - f , the data (mean ± SEM) are representative of three independent experiments. Each dot represents a biological replicate (n = 3). The p values were calculated by two-tailed Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.001, NS-not significant.

Article Snippet: For mouse cGAS catalytic domain, 2.5 μM protein was incubated with 0.2 mg/ml salmon sperm DNA (Invitrogen, 15632-011) for 1 h at 37 °C.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Agarose Gel Electrophoresis, Luciferase, Transfection, Plasmid Preparation, Control, Two Tailed Test

Journal: Nature

Article Title: The Molecular Basis of Tight Nuclear Tethering and Inactivation of cGAS

doi: 10.1038/s41586-020-2749-z

Figure Lengend Snippet: a. Agarose gel shift assay shows that mutations at the nucleosome binding surface of human cGAS affect the binding of a 45 bp dsDNA. b. Enzyme activity assays of mouse cGAS catalytic domain mutants by ion exchange chromatography. In this assay, 2.5 μM mcGAS was incubated with 0.2 mg/ml salmon sperm DNA. WT mcGAS and negative control without DNA are colored in green and blue. Mutations that abolish nucleosome binding are colored red. The negative control mutation K382E is colored orange. c. Enzyme activity assays of full-length human cGAS mutants by ion exchange chromatography. In this assay, 2.5 μM hcGAS was incubated with 0.2 mg/ml salmon sperm DNA. WT hcGAS and negative control without DNA are colored in green and blue. Mutations that abolish nucleosome binding are colored red. The negative control mutation K394E is colored orange. d. IFN-β luciferase reporter assays show that mutations of hcGAS affect signaling in HEK 293T cells. Luciferase reporter signals from the cells transfected with 0.025 ng cGAS and 0.4 ng STING are indicated by the orange bars, from cells transfected with 0.00625 ng cGAS and 0.4 ng STING by the cyan bars, from cells transfected with 0.025 ng cGAS, 0.4 ng STING, or the vector control by the green, brown and purple bars, respectively. The data (mean ± SEM) are representative of three independent experiments. Each dot represents a biological replicate (n = 3). The p values were calculated by two-tailed Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.001, NS-not significant. e. Western blot shows that WT mouse cGAS and its mutants have similar expression level in the transfected HEK 293T cells. f. Western blot shows WT human cGAS and its mutants have similar expression level in the transfected HEK 293T cells.

Article Snippet: For mouse cGAS catalytic domain, 2.5 μM protein was incubated with 0.2 mg/ml salmon sperm DNA (Invitrogen, 15632-011) for 1 h at 37 °C.

Techniques: Agarose Gel Electrophoresis, Shift Assay, Binding Assay, Activity Assay, Ion Exchange Chromatography, Incubation, Negative Control, Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Control, Two Tailed Test, Western Blot, Expressing